Methods and compositions for targeted protein degradation

ABSTRACT

Coronatine has been found to enhance binding of the JAZ1 degron to the  Arabidopsis  F-box protein COI1, and analysis of the JAZ1 degron sequence has resulted in the identification of specific peptide sequences that bind COI1 with high affinity in the presence of coronatine. Crystal structure analysis has determined that coronatine and JA-Ile enhance the interaction between COI1 and JAZ1 via binding to a specific binding pocket on COI1. Attachment of one or more JAZ1 peptide tags as disclosed herein to a target protein in a non-plant cell expressing  Arabidopsis  COI1 or a homolog thereof results in degradation of the target protein following addition of a molecule that binds the coronatine/JA-Ile binding pocket on COI1. Therefore, provided herein are compositions, methods, and kits for targeted protein degradation.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication No. 61/352,758, filed Jun. 8, 2010, the disclosure of whichis incorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under grant number2RO1CA107134 awarded by National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Reverse genetic approaches are a powerful laboratory tool fordetermining the function of a target protein. The target protein is“knocked down,” and cellular changes are observed in order to infer thenormal function of the knocked down target. Methods for knocking down atarget protein by manipulating DNA transcription and RNA translation arewell established. Among the most commonly used are gene knockout inwhole animals and degradation of target mRNA using siRNA and shRNAtechniques. However, delivery of RNA molecules can be cumbersome, andthese methods often cannot achieve 100% efficiency.

Although several methods exist for knocking down a protein target at thetranscription and translation levels, there are very few options forknocking down a protein target once the protein has been made. Suchmethods are desirable because most small molecule therapeutics operateby manipulating proteins directly. Therefore, targeted degradationtechniques that operate at the protein level provide the best tool forexamining the potential effects of small molecule therapeutics.

The ideal protein knock down system would function at the protein level,and would be capable of tightly controlling protein levels in a temporalmanner. Temporal control of protein levels allows down-regulation ofproteins that are essential for the full development of a system orpathway, and can be used to study dynamic biological functions which areotherwise difficult to manipulate.

SUMMARY

In certain embodiments, methods are provided for targeted proteindegradation. In certain of these embodiments, the protein targeted fordegradation is tagged with one or more JAZ peptide tags as providedherein. The target protein is expressed in a non-plant host cell thatalso expresses the Arabidopsis protein COI1 or a homolog thereof, and amolecule that binds the COI1/JA-Ile binding pocket of COI1 is introducedinto the cell to induce target protein degradation. In certainembodiments, the peptide tag comprises, consists of, or consistsessentially of an amino acid sequence as set forth in SEQ ID NOs: 5, 6,7, or 13. In certain embodiments, the molecule that binds theCOI1/JA-Ile binding pocket of COI1 is coronatine, JA, or a JA amino acidconjugate such as JA-Ile. In certain embodiments, an inositolpentakisphosphate cofactor may also be introduced into the cell. Incertain embodiments, the non-plant host cell may be a eukaryotic cellsuch as a yeast or mammalian cell. In certain embodiments, theArabidopsis protein COI1 or a homolog thereof may be COI1 fromArabidopsis thaliana or Arabidopsis lyrata, or it may be a COI1 homologfrom another plant or moss such as rice, tomato, grape, poplar, castoroil, corn, rubber tree, pea, wild tobacco, soybean, sorghum, wheat, orPhyscomitrella patens. The target protein may be either an endogenoushost cell protein or an exogenous protein. In those embodiments whereinthe target protein is an endogenous host cell protein, the peptide tagmay be attached to the target protein by introducing a DNA sequenceencoding the peptide tag adjacent to the DNA sequence encoding thetarget protein in the host cell, such that the target protein isexpressed with the peptide tag attached. In those embodiments whereinthe target protein is an exogenous protein, the target protein may beintroduced into the host cell via a DNA sequence encoding the targetprotein and the peptide tag. In certain embodiments, target proteindegradation may be halted by deactivating the molecule that binds theCOI1/JA-Ile binding pocket of COI1 or removing it from the cell. Inother embodiments, target protein degradation may be halted by naturaldegradation of the molecule that binds the COI1/JA-Ile binding pocket ofCOI1.

In certain embodiments, methods are provided for targeted proteindegradation. In certain of these embodiments, the protein targeted fordegradation is tagged with one or more peptide tags comprising an aminoacid sequence as set forth in SEQ ID NOs:5, 6, 7, or 13. The targetprotein is expressed in a non-plant host cell that also expresses theArabidopsis protein COI1 or a homolog thereof, and coronatine or JA-Ileis introduced into the cell to induce target protein degradation. Incertain embodiments, an inositol pentakisphosphate cofactor may also beintroduced into the cell. In certain embodiments, the non-plant hostcell may be a eukaryotic cell such as a yeast or mammalian cell. Incertain embodiments, the Arabidopsis protein COI1 or a homolog thereofmay be COI1 from Arabidopsis thaliana or Arabidopsis lyrata, or it maybe a COI1 homolog from another plant or moss such as rice, tomato,grape, poplar, castor oil, corn, rubber tree, pea, wild tobacco,soybean, sorghum, wheat, or Physcomitrella patens. The target proteinmay be either an endogenous host cell protein or an exogenous protein.In those embodiments wherein the target protein is an endogenous hostcell protein, the peptide tag may be attached to the target protein byintroducing a DNA sequence encoding the peptide tag adjacent to the DNAsequence encoding the target protein in the host cell, such that thetarget protein is expressed with the peptide tag attached. In thoseembodiments wherein the target protein is an exogenous protein, thetarget protein may be introduced into the host cell via a DNA sequenceencoding the target protein and the peptide tag. In certain embodiments,target protein degradation may be halted by deactivating coronatine orJA-Ile or removing them from the cell. In other embodiments, targetprotein degradation may be halted by natural degradation of coronatineor JA-Ile.

In certain embodiments, methods are provided for targeted proteindegradation in a non-plant host cell by fusing a target protein to apeptide tag as provided herein, introducing a DNA sequence encodingArabidopsis COI1 or a homolog thereof into the host cell, culturing thehost cell under conditions that result in the expression of the targetprotein and Arabidopsis COI1 or a homolog thereof, and introducing amolecule that binds the COI1/JA-Ile binding pocket of COI1 into the hostcell. In certain embodiments, the peptide tag comprises, consists of, orconsists essentially of an amino acid sequence as set forth in SEQ IDNOs:5, 6, 7, or 13. In certain embodiments, the molecule that binds theCOI1/JA-Ile binding pocket of COI1 is coronatine, JA, or a JA amino acidconjugate such as JA-Ile. In certain embodiments, an inositolpentakisphosphate cofactor may also be introduced into the cell. Incertain embodiments, the non-plant host cell may be a eukaryotic cellsuch as a yeast or mammalian cell. In certain embodiments, theArabidopsis protein COI1 or a homolog thereof may be COI1 fromArabidopsis thaliana or Arabidopsis lyrata, or it may be a COI1 homologfrom another plant or moss such as rice, tomato, grape, poplar, castoroil, corn, rubber tree, pea, wild tobacco, soybean, sorghum, wheat, orPhyscomitrella patens. The target protein may be either an endogenoushost cell protein or an exogenous protein. In those embodiments whereinthe target protein is an endogenous host cell protein, the peptide tagmay be attached to the target protein by introducing a DNA sequenceencoding the peptide tag adjacent to the DNA sequence encoding thetarget protein in the host cell, such that the target protein isexpressed with the peptide tag attached. In those embodiments whereinthe target protein is an exogenous protein, the target protein may beintroduced into the host cell via a DNA sequence encoding the targetprotein and the peptide tag. In certain embodiments, target proteindegradation may be halted by deactivating the molecule that binds theCOI1/JA-Ile binding pocket of COI1 or removing it from the cell. Inother embodiments, target protein degradation may be halted by naturaldegradation of the molecule that binds the COI1/JA-Ile binding pocket ofCOI1.

In certain embodiments, methods are provided for targeted proteindegradation in a non-plant host cell by fusing a target protein to apeptide tag comprising, consisting of, or consisting essentially of anamino acid sequence as set forth in SEQ ID NOs:5, 6, 7, or 13,introducing a DNA sequence encoding Arabidopsis COI1 or a homologthereof into the host cell, culturing the host cell under conditionsthat result in the expression of the target protein and Arabidopsis COI1or a homolog thereof, and introducing coronatine or JA-Ile into the hostcell. In certain embodiments, an inositol pentakisphosphate cofactor mayalso be introduced into the cell. In certain embodiments, the non-planthost cell may be a eukaryotic cell such as a yeast or mammalian cell. Incertain embodiments, the Arabidopsis protein COI1 or a homolog thereofmay be COI1 from Arabidopsis thaliana or Arabidopsis lyrata, or it maybe a COI1 homolog from another plant or moss such as rice, tomato,grape, poplar, castor oil, corn, rubber tree, pea, wild tobacco,soybean, sorghum, wheat, or Physcomitrella patens. The target proteinmay be either an endogenous host cell protein or an exogenous protein.In those embodiments wherein the target protein is an endogenous hostcell protein, the peptide tag may be attached to the target protein byintroducing a DNA sequence encoding the peptide tag adjacent to the DNAsequence encoding the target protein in the host cell, such that thetarget protein is expressed with the peptide tag attached. In thoseembodiments wherein the target protein is an exogenous protein, thetarget protein may be introduced into the host cell via a DNA sequenceencoding the target protein and the peptide tag. In certain embodiments,target protein degradation may be halted by deactivating coronatine orremoving it from the cell. In other embodiments, target proteindegradation may be halted by natural degradation of coronatine.

In certain embodiments, methods are provided for targeted proteindegradation in a host animal by introducing a DNA sequence encoding thetarget protein linked to a peptide tag as provided herein and anotherDNA sequence encoding Arabidopsis COI1 or a homolog thereof, expressingthe tagged target protein and COI1, and then administering a moleculethat binds the COI1/JA-Ile binding pocket of COI1 to the animal. Incertain embodiments, an inositol pentakisphosphate cofactor may also beintroduced into the cell. In certain embodiments, the peptide tagcomprises, consists of, or consists essentially of an amino acidsequence as set forth in SEQ ID NOs:5, 6, 7, or 13. In certainembodiments, the molecule that binds the COI1/JA-Ile binding pocket ofCOI1 is coronatine, JA, or a JA amino acid conjugate such as JA-Ile. Incertain embodiments, the animal is a mammal, and in certain of theseembodiments the animal is a mouse. In certain embodiments, theArabidopsis protein COI1 or a homolog thereof may be COI1 fromArabidopsis thaliana or Arabidopsis lyrata, or it may be a COI1 homologfrom another plant or moss such as rice, tomato, grape, poplar, castoroil, corn, rubber tree, pea, wild tobacco, soybean, sorghum, wheat, orPhyscomitrella patens.

In certain embodiments, methods are provided for targeted proteindegradation in a host animal by introducing a DNA sequence encoding thetarget protein linked to a peptide tag comprising, consisting of, orconsisting essentially of an amino acid sequence as set forth in SEQ IDNOs:5, 6, 7, or 13 and another DNA sequence encoding Arabidopsis COI1 ora homolog thereof, expressing the tagged target protein and COI1, andthen administering coronatine or JA-Ile to the animal. In certainembodiments, an inositol pentakisphosphate cofactor may also beintroduced into the cell. In certain embodiments, the animal is amammal, and in certain of these embodiments the animal is a mouse. Incertain embodiments, the Arabidopsis protein COI1 or a homolog thereofmay be COI1 from Arabidopsis thaliana or Arabidopsis lyrata, or it maybe a COI1 homolog from another plant or moss such as rice, tomato,grape, poplar, castor oil, corn, rubber tree, pea, wild tobacco,soybean, sorghum, wheat, or Physcomitrella patens.

In certain embodiments, non-plant host cells are provided that comprisea DNA sequence encoding a target protein linked to a peptide tagcomprising, consisting of, or consisting essentially of an amino acidsequence as set forth in SEQ ID NOs:5, 6, 7, or 13 and another DNAsequence encoding Arabidopsis COI1 or a homolog thereof. In certainembodiments, the cells further comprise an inositol pentakisphosphatecofactor. In certain embodiments, the non-plant host cell is aeukaryotic cell, and in certain of these embodiments the non-plant hostcell is a yeast or mammalian cell. In certain embodiments, theArabidopsis protein COI1 or a homolog thereof may be COI1 fromArabidopsis thaliana or Arabidopsis lyrata, or it may be a COI1 homologfrom another plant or moss such as rice, tomato, grape, poplar, castoroil, corn, rubber tree, pea, wild tobacco, soybean, sorghum, wheat, orPhyscomitrella.

In certain embodiments, methods are provided for targeting an endogenoustarget protein in a non-plant host cell for coronatine- orJA-Ile-induced degradation by introducing a DNA sequence encoding apeptide tag comprising, consisting of, or consisting essentially of anamino acid sequence as set forth in SEQ ID NOs:5, 6, 7, or 13 such thatthe DNA sequence is inserted adjacent to the gene encoding theendogenous target protein, and such that the target protein is expressedfused to the peptide tag.

In certain embodiments, peptides are provided for tagging a targetprotein for degradation, and in certain of these embodiments, thepeptide tags comprise, consist of, or consist essentially of an aminoacid sequence as set forth in SEQ ID NOs:5, 6, 7, or 13. Also providedin certain embodiments are isolated nucleic acid sequences encodingthese peptide tags, as well as the use of the peptide tags in taggingtarget proteins for degradation.

In certain embodiments, kits are provided for targeted proteindegradation. In certain embodiments, these kits may include one or moreof the following: an isolated nucleic acid encoding a peptide tag asprovided herein, an isolated nucleic acid encoding Arabidopsis COI1 or ahomolog thereof, and a molecule that binds the COI1/JA-Ile bindingpocket of COI1. In certain embodiments, the kit may further comprise atarget protein or an isolated nucleic acid encoding a target protein,and/or an inositol pentakisphosphate cofactor. In certain embodiments,the peptide tag comprises, consists of, or consists essentially of anamino acid sequence as set forth in SEQ ID NOs:5, 6, 7, or 13. Incertain embodiments, the molecule that binds the COI1/JA-Ile bindingpocket of COI1 is coronatine, JA, or a JA amino acid conjugate such asJA-Ile. In certain embodiments, the kit further comprises instructionsfor use and/or other printed materials.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: A. Binding of ³H-coronatine (300 nM) to COI1 alone, JAZ6 alone,JAZ6/COI1, JAZ1/COI1, and JAZ6F195A/COI1. B. Saturation binding of³H-coronatine to the complex of COI1/ASK1 in the presence of JAZ1 (▴, KDof 68±15 nM) and JAZ6 (▪, KD of 48±13 nM).

FIG. 2: A. Saturation binding of ³H-coronatine (up to 3 μM) to theCOI1/JAZ6 complex, isolated COI1, and isolated JAZ6. B. Competitionbinding of 100 nM ³H—COR with (3R,7S)-JA-Ile and (3R,7R)-JA-Ile at aK_(i) of 1.8±0.6 μM and 18±19 μM, respectively.

FIG. 3: Binding of 300 nM ³H—COR to JAZ1 proteins truncated immediatelyafter (Δ227-253) or before (Δ224-253) the PY motif.

FIG. 4: Binding of 300 nM coronatine to COI1 in the presence of variousJAZ1 degron derivative peptides with systematic N-terminal extensions.

FIG. 5: Saturation binding of COI1/ASK1 and the JAZ1 degron +5 peptide.The peptide bound COI1 with a K_(D) of 108±29 nM.

FIG. 6: Binding of coronatine to COI1 in the presence of various JAZ1degron derivative peptides.

FIG. 7: A, B. Structure of Arabidopsis COI1 (green ribbon)/Ask1 receptorprotein (grey ribbon) complex bound to JAZ1 degron peptide (orangeribbon) and (3R,7S)-JA-Ile in yellow space fill representation. C.Surface representation of COI1 (grey) with loop 2 (blue), loop 12(purple), and loop 14 (green) forming the JA-Ile binding pocket.

FIG. 8: A, B. Side view of JA-Ile and COR binding. Hormones are shown asstick models, along with positive F_(o)-F_(c) electron density,calculated before they were built into the model (red mesh). Hydrogenbond and salt bridge networks are shown with yellow dashes. C. Top viewof the JA-Ile pocket showing the F_(o)-F_(c) electron density,calculated before JA-Ile was built into the model (red mesh). Theelectron density of the pentenyl side chain of (3R,7S)-JA-Ile cannotaccommodate the (3R,7R)-JA-Ile side chain, which is constrained by thechiral configuration at the C7 position.

FIG. 9: Alignment of Arabidopsis TIR1 and various COI1 orthologs fromselect plant species. Secondary structure elements as determined in thecrystal structure of the COI1/ASK1/JAZ1 degron peptide/JA-Ile complexare shown on top of the Arabidopsis thaliana COI1 sequence. Criticalligand-, phosphate-, and substrate-contacting residues are indicated bycolored dots as described in the key.

FIG. 10: When bound to COI1, JA-Ile (yellow space fill) is solventaccessible at both the keto group (top) and carboxyl group (bottom).

FIG. 11: A. Side view of the COI1 pocket accommodating the pentenyl sidechain of (3R,7S)-JA-Ile (yellow stick). The pentenyl side chain of(3R,7R)-JA-Ile (magenta stick) is modeled on the structure of(3R,7S)-JA-Ile and rotated around the C7-C8 bond to minimize collisionwith JAZ1 Ala 204 and COI1 Phe 89. The electron clouds of nearby COI1(green) and JAZ1 (orange) side chains, as well as the pentenyl sidechain of (3R,7R)-JA-Ile (magenta) are shown in dot form. Ala 86 and Leu91 of COI1 blocking the front view of the pocket are omitted forclarity. B. Side view of (3R,7S)-JA-Ile (yellow stick) and coronatine(cyan stick) showing a hydrophobic pocket that accommodates both thealiphatic isoleucine portion of JA-Ile and the cyclopropane ring ofcoronatine.

FIG. 12: A. Top view of the complete JAZ1 degron peptide (orange) boundto COI1 (green) and JA-Ile (yellow). B. Side view and surfacerepresentation of the JAZ peptide, which acts as a clamp to lock JA-Ilein the pocket.

FIG. 13:A. Interactions of the N-terminal region of the JAZ1 degron withCOI1 and JA-Ile. Hydrogen bonds are shown with yellow dashes. B.Structural role of the Arg 206 residue from the JAZ1 degron incoordinating the carboxyl group of JA-Ile with three basic residues ofthe COI1 ligand pocket floor. C. Top view of the amphipathic JAZ1 degronhelix bound to COI1 with three hydrophobic residues of JAZ1 shown instick representation (orange) and COI1 residues in colored surfacerepresentation.

FIG. 14: A. Binding assays performed with 100 nM ³H-coronatine, dialyzedCOI1, and 1 μM synthetic InsP₅. B. Saturation binding of ³H-coronatineto dialyzed COI1 in the presence of 1 μM of InsP₅ and InsP₆ at a K_(d)of 30±5 nM and 37±8 nM, respectively. All results are the mean±s.e. ofup to three experiments performed in duplicate.

FIG. 15: Nano-electrospray mass spectrometry of the intact COI/ASK1complex. Low-intensity charge series corresponds in mass to thecofactor-free COI1/ASK1 complex.

FIG. 16: Structural mass spectrometry analysis of the COI1/ASK1 complex.A. Isolation at 4564 m/z of the 19+ charge state for tandem MS analysis(shown in blue in FIG. 24). B. MS/MS spectrum showing the dissociationproducts of ions isolated at 4588 m/z (shown in orange in FIG. 24).

FIG. 17: Optimized cofactor purification scheme.

FIG. 18: Proton TOCSY spectrum of the purified cofactor. Numbers alongthe diagonal indicate the positions of the six protons ofIns(1,2,4,5,6)P₅. The cross-peaks corresponding to direct couplings arelabeled. Other cross-peaks correspond to relayed connectivities.

FIG. 19: TOCSY spectrum of a synthetic Ins(1,2,4,5,6)P₅ as a standard.

FIG. 20: Mass spectrometry analysis of Ins(1,2,4,5,6)P₅ purified fromrecombinant COI1/ASK1.

FIG. 21: A. Islands of positive F_(o)-F_(c) electron density (red mesh)below the hormone-binding pockets, which probably belong to inorganicphosphate molecules from the crystallization solutions that displaceInsP₅ from the InsP₅-binding site. B. Bottom view of a surfaceelectrostatic potential representation of COI1 from positive (blue) tonegative (red).

FIG. 22: Surface conservation mapping of COI1. Conservation mapping ofCOI1 surface based on sequences of COI1 orthologs from nine differentspecies (A. thaliana, H. brasiliensis, R. communis, P. trichocarpa, V.cinifera, P. sativum, S. lycopersicum, Z. mays, O. sativa). Dark blue,light blue, and white surface regions indicate 98-100%, 60-98%, and <60%sequence conservation, respectively. The F-box portion of COI1 and itsassociated ASK1 are carved out for clarity reasons. Four phosphatemolecules bound to COI1 are shown by red sticks. JAZ1 peptide and ASK1are shown in grey.

FIG. 23: A. Binding of ³H-coronatine at 100 nM to a complex of COI1 andJAZ1, with the addition of 1 μM synthetic Ins(1,2,4,5,6)P₅ (InsP₅). B.With extensive dialysis to remove the co-purified InsP₅ cofactor, 100 nM³H-coronatine no longer binds dialyzed COI1 in the presence of JAZ1.Synthetic InsP₅ rescues binding. C. InsP₅ rescues the binding of 100 nM³H-coronatine to dialyzed COI1/ASK1 in the presence of JAZ1 with an EC₅₀of 27±12 nM.

FIG. 24: Interwoven hydrogen bond network in the complex structure.

FIG. 25: A. Close-up view of COI1 residues (green stick) in closevicinity to the inorganic phosphates occupying the InsP5 binding pocket(orange stick, with along with positive F_(o)-F_(c) density in redmesh). Hydrogen bonds are shown with yellow dashes. B. Interaction ofwild-type COI1 and COI1 mutants with JAZ1 detected by yeast two-hybridassay.

DETAILED DESCRIPTION

The following description of the invention is merely intended toillustrate various embodiments of the invention. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to one skilled in the artthat various equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein.

The following abbreviations are used herein: COR, coronatine; cpm,counts per minute; JA, jasmonic acid; JA-Ile, jasmonyl-L-isoleucine;JAZ, jasmonate ZIM-domain; LRR, leucine-rich repeat; ppm, parts permillion; SCF, Skp1-Cullin-F box protein; sdm, site-directed mutants.

There are currently no satisfactory methods for knocking down a proteintarget in a temporally controlled manner using targeted proteindegradation. Several previous attempts at developing such a method haveutilized the ubiquitin proteasome system. However, all of these methodshave serious drawbacks.

One method that employs the ubiquitin proteasome system utilizespeptide-small molecule hybrids (“protacs”). These chimeric moleculesencourage binding of a target protein to the SCF^(βTrCP) complex,resulting in proteolysis of the target (Sakamoto 2000). However, theprotacs used in these methods are not membrane-permeable, and thereforerequire modifications to increase cell permeability or the use ofmicroinjection. Another disadvantage of this system is off-targeteffects that arise from “swamping out” SCF^(βTrCP) and blocking itsability to interact with endogenous targets. Thus, modification ofendogenous SCF complexes has shown very limited usefulness as a reversegenetics tool. Another previously developed method for targeted proteindegradation utilized chimeric fusions consisting of the target proteinand large binding domains of proteins targeted for degradation at SCFcomplexes. One such system paired the target protein with the chiretinoblastoma (Rb)-binding domain derived from the human papillomavirus(HPV) oncoprotein E7 (Zhou 2000). This method showed some success inyeast and mammalian systems, but it is not inducible and requires theuse of an obstructive large tag. A similar chimeric approach requiredengineering of the F-box protein β-TrCP with a small, phosphorylatedpeptide that encourages binding of a particular target molecule to theF-box protein (Zhang 2003). However, this system required a great dealof engineering in order for the chimeric F-box protein to recognize onlythe target of interest and not endogenous targets. Therefore, it has notbeen widely used.

Recently, researchers in Japan have developed a method that utilizes theArabidopsis and tomato auxin receptor TIR1 for rapid, hormone-inducedprotein degradation (Nishimura Nature Methods 2009). TIR1 integratesinto the yeast and mammalian SCF scaffold and degrades proteins with theAUX/IAA tag, the natural substrate of TIR1. However, this tag is over 20kDa, a large tag by biochemistry standards. A smaller tag has not yetbeen identified. As such, the TIR1 system has limited usefulness.

The phytohormone jasmonic acid (JA) and its metabolites regulate a widespectrum of plant physiology, participating in normal development andgrowth processes as well as defense responses to environmental andpathogenic stressors. JA is activated upon specific conjugation to theamino acid L-isoleucine, which produces the highly bioactive hormonalsignal (3R,7S)-jasmonyl-L-isoleucine (JA-Ile). Coronatine (COR) is aPseudomonas syringae virulence factor that structurally mimics JA-Ile.

The discovery of coronatine-insensitive mutants enabled theidentification of COI1 as a key player in the JA pathway. ArabidopsisCOI1 is an F-box protein that functions as the substrate-recruitingmodule of the Skp1-Cullin-F box protein (SCF) ubiquitin E3 ligasecomplex. Like other E3 ligases, SCF^(CO1) is involved in theubiquitination of proteins, which targets the proteins for subsequentdegradation by the 26S proteasome.

Arabidopsis jasmonate ZIM-domain (JAZ) proteins such as JAZ1, JAZ6,JAZ7, and JAZ8 are SCF^(CO1) substrate targets that associate with COI1in a hormone-dependent manner. In the absence of hormone signal, the JAZproteins actively repress the transcription factor MYC2, which binds tocis-acting elements of jasmonate-response genes. In response to cuesthat upregulate JA-Ile synthesis, the hormone stimulates the specificbinding of JAZ proteins to COI1, leading to poly-ubiquitylation andsubsequent degradation of the JAZ proteins by the 26S proteasome. JAZdegradation relieves repression of MYC2 and probably other transcriptionfactors, permitting the expression of JA-responsive genes. The role ofCOI1-mediated JAZ degradation in JA signaling is analogous to auxinsignaling through the F-box protein TIR1, which promoteshormone-dependent turnover of the AUX/IAA transcriptional repressors.

The experimental results provided herein disclose the identification andcharacterization of a complete jasmonate receptor comprising COI1, a JAZpeptide, and inositol pentakisphosphate. Coronatine was found to bindthe complexes of COI1/JAZ1 and COI1/JAZ6 complexes with high affinitywhile displaying minimal binding affinity for COI1, JAZ1, or JAZ6 alone.Crystal structure studies were used to identify a coronatine- andJA-Ile-binding pocket on COI1. Binding of these molecules to the COI1binding pocket increases binding affinity between COI1 and JAZ1.Coronatine has been found to play a similar role in the binding of JAZ2,JAZ3, JAZ4, JAZ5, JAZ6, JAZ8, JAZ9, JAZ10, JAZ11, and JAZ12 to COI1. Asingle isoform of inositol pentakisphosphate (Ins(1,2,4,5,6)P₅) wasfound to co-purify with COI1, and functional assays showed that thismolecule is a critical cofactor in the interaction between COI1 and JAZproteins.

The COI1 binding region of JAZ proteins had previously been mapped to acarboxy-terminal Jas motif. To precisely map the minimal region of theJas motif that it is required for high affinity binding of COI1 to JAZ1in the presence of coronatine, the JAZ1 degron and various derivativesthereof were analyzed. This led to the identification of specific JAZpeptide tags with enhanced binding affinity for COI1 in the presence ofcoronatine.

Provided herein in certain embodiments are compositions comprising oneor more JAZ peptide tags capable of binding Arabidopsis COI1 or ahomolog thereof, as well as nucleic acids encoding these JAZ peptidetags, methods of using these peptide tags to mark a target protein fordegradation, and the use of these peptide tags in various methods andkits for temporally controlled protein degradation.

In certain embodiments, the JAZ peptide tags provided herein consist of,consist essentially of, or comprise the JAZ1 degron as set forth in SEQID NO: 1. In other embodiments, the peptide tags consist of, consistessentially of, or comprise an amino acid sequence that corresponds tothe JAZ1 degron of SEQ ID NO:1 but with one or more additions,deletions, or substitutions. In certain of these embodiments, thepeptide tags consist of, consist essentially of, or comprise the aminoacid sequence of SEQ ID NO: 1 but with one or more deletions from theC-terminal end. In certain other of these embodiments, the peptide tagsconsist of, consist essentially of, or comprise the amino acid sequenceof SEQ ID NO:1 but with one or more additions to the N-terminal end. Inother embodiments, the peptide tags provided herein comprise a fragmentof an Arabidopsis JAZ protein other than JAZ1, such as for example JAZ2,JAZ3, JAZ4, JAZ5, JAZ6, JAZ8, JAZ9, JAZ10, JAZ11, or JAZ12.

In certain preferred embodiments, the JAZ1 peptide tags disclosed hereinconsist of, consist essentially of, or comprise the amino acid sequenceof SEQ ID NOs:5, 6, or 7. In other preferred embodiments, the peptidetags consist of, consist essentially of, or comprise an amino acidsequence that corresponds to the amino acid sequences of SEQ ID NOs:5,6, or 7, but wherein one or more amino acid substitutions, additions, ordeletions have been introduced into the sequence. For example, incertain embodiments the peptide tags may consist of, consist essentiallyof, or comprise the amino acid sequenceX₁X₂X₃X₄X₅RRX₈SLHRFLEKRKDRVX₂₂X₂₃X₂₄X₂₅X₂₆X₂₇ (SEQ ID NO: 13), whereinX₁, X₂, X₃, X₄, and X₅ are each independently absent or any amino acid,Xs is either Ala or Lys, and X₂₂, X₂₃, X₂₄, X₂₅, X₂₆, and X₂₇ are eachindependently absent or any amino acid. In certain of these embodiments,X₁ is either absent, Glu, or Val; X₂ is either absent, Leu, or Glu; X₃is either absent, Pro, or Arg; X₄ is either absent or Ile; and X₅ iseither absent or Ala. In certain embodiments, X₂₂ is either absent orThr; X₂₃ is either absent or Ser; X₂₄ is either absent or Lys; X₂₅ iseither absent or Ala; X₂₆ is either absent or Pro; and X₂₇ is eitherabsent or Tyr.

The small, unobtrusive peptide tags provided herein are superior to thelarge chimeric tags used in previously developed methods for targetedprotein destruction because they are less likely to interfere withprotein function, complex formation, and subcellular localization.However, in certain embodiments, the peptide tags disclosed herein maycomprise a longer sequence, such as for example the full-length JAZ1polypeptide sequence as set forth in SEQ ID NO: 14 or the full-lengthJAZ10 polypeptide sequence set forth in SEQ ID NO:29. In other of theseembodiments, the peptide tag may comprise the full-length JAZ2, JAZ3,JAZ4, JAZ5, JAZ6, JAZ8, JAZ9, JAZ11, or JAZ12 polypeptide sequence.

COI1 binding to a protein comprising a JAZ peptide tag as results indegradation of the protein. As disclosed herein, binding of coronatineor JA-Ile to a specific binding pocket in COI1 enhances the interactionof COI1 and the JAZ peptide. Thus, targeted protein degradation can beaccomplished by tagging a target protein with a JAZ peptide tag asprovided herein, then contacting the protein with COI1 in the presenceof a molecule that binds to the coronatine/JA-Ile binding pocket ofCOI1.

As such, provided herein in certain embodiments are methods for targetedprotein degradation that utilize one or more of 1) Arabidopsis COI1 or ahomolog thereof, 2) a molecule that binds the COI1/JA-Ile binding pocketin COI1, 3) one or more JAZ peptide tags, and, optionally, 4) aninositol pentakisphosphate cofactor. Also provided herein arecompositions and kits for carrying out these methods.

The crystal structure analysis provided herein shows that coronatine andJA-Ile interact with a specific set of residues in the COI1 bindingpocket that includes R85, A86, F89, L91, R348, E350, A384, Y386, R409,V411, Y444, LA69, R496, and W519 of SEQ ID NO:15. Therefore, a “moleculethat binds the COI1/JA-Ile binding pocket of COI1” as used herein refersto a molecule that interacts with one or more of these residues, andmore preferably with all fourteen of these residues. As used herein, amolecule “interacts” with a particular COI1 binding pocket residue if,when the molecule is bound to COI1, any portion of the molecule resideswithin a molecular distance that is within the hydrogen bond or Van derWaals interaction radius (approximately 2.5 to 4 Å) of the residue. Incertain embodiments of the compositions, methods, and kits providedherein, a molecule that binds the COI1/JA-Ile binding pocket of COI1 iscoronatine. In other embodiments, the molecule is JA or a JA-amino acidconjugate such as JA-Ile, JA-L-leucine, JA-L-valine, or JA-L-alanine.

In those embodiments of the methods, compositions, and kits providedherein that utilize or comprise an inositol pentakisphosphate cofactor,the inositol pentakisphosphate cofactor may beinositol-1,2,4,5,6-pentakisphosphate. In other embodiments, the cofactormay be another molecule of the myo-inositol family, such asinositol-1,4,5,6-tetrakisphosphate.

In certain embodiments of the methods provided herein, one or more JAZpeptide tags are attached to or incorporated into a target protein. Inthe presence of molecule that binds the COI1/JA-Ile binding pocket ofCOI1, the JAZ peptide tag (and hence the target protein) binds toArabidopsis COI1 or a homolog thereof with high affinity, resulting intarget protein degradation.

Unlike previously developed methods for targeted protein destruction,the methods provided herein utilize small molecules that aremembrane-permeable and require minimal engineering. The methods providedherein are rapidly inducible and easily reversible, and they do notrequire the use of large, obstructive tags. Thus, these methodsrepresent a cheap, simple means for targeted protein destruction in vivothat is significantly superior to previously developed methods.

In certain embodiments of the methods disclosed herein, the targetprotein is tagged with one or more JAZ peptide tags as disclosed herein.JAZ peptide tags may be attached to a target protein using any methodsknown in the art. For example, in certain embodiments the peptide tagmay be attached to the target protein prior to target proteinexpression. In these embodiments, a DNA sequence encoding the peptidetag is introduced into the host cell adjacent to the DNA sequenceencoding the target protein, such that the peptide tag is expressed aspart of the target protein. Methods for introducing a DNA sequenceencoding a peptide tag into a cell and expressing a protein attached tothe peptide tag are well known in the art. In other embodiments, thepeptide tag may be attached to the target protein after the targetprotein has been expressed.

In certain embodiments of the methods disclosed herein, the targetprotein is tagged with a single peptide tag. In other embodiments, thetarget protein is tagged with two or more peptide tags. In thoseembodiments wherein the target protein is tagged with two or morepeptide tags, the peptide tags may have the same or different sequences.

Previously developed targeted protein destruction systems have generallyutilized chimeric mammalian F-box proteins. However, the methodsdisclosed herein utilize the Arabidopsis F-box protein COI1 or plant ormoss homologs thereof. In certain embodiments, the methods disclosedherein utilize Arabidopsis thaliana or Arabidopsis lyrata COI1comprising the amino acid sequence set forth in SEQ ID NOs: 15 and 16,respectively. Specific plant or moss homologs that may be utilizedinclude those from rice (Oryza sativa, SEQ ID NO:17), tomato (Solanumlycopersicum, SEQ ID NO:18), grape (Vitis vinifera, SEQ ID NO:19),poplar (Populus trichocarpa, SEQ ID NO:20 and SEQ ID NO:21), castor oil(Ricinis communis, SEQ ID NO:22), corn (Zea mays, SEQ ID NO:23), rubbertree (Hevea brasiliensis, SEQ ID NO:24), pea (Pisum sativum, SEQ IDNO:25), wild tobacco (Nicotiana attenuata, SEQ ID NO:26), soybean(Glycine max, SEQ ID NO:27), sorghum (Sorghum bicolor, SEQ ID NO:28),wheat, or Physcomitrella patens. COI1 and its homologs are preferable tomammalian proteins because their high specificity for plant substratesminimizes competition and unwanted degradation of endogenous targets.

In certain embodiments, an Arabidopsis COI1 polypeptide or a homologthereof may be introduced directly into a cell. In other embodiments,one or more genes encoding Arabidopsis COI1 or a homolog thereof may beintroduced into a cell such that the cell expresses Arabidopsis COI1 ora homolog thereof. In these embodiments, the one or more genes encodingArabidopsis COI1 or a homolog thereof may be introduced into a cell viaany method known in the art, including transformation or transient orstable transfection using viral and non-viral vectors. In certainembodiments, the one or more genes may be introduced using a viralvector such as an adenoviral, retroviral, lentiviral, or baculoviralvector. Provided herein in certain embodiments are viral and non-viralvectors comprising a DNA sequence encoding Arabidopsis COI1 or a homologthereof, as well as methods of using these vectors to achieve expressionof Arabidopsis COI1 or a homolog thereof in a non-plant host cell. Incertain embodiments, a DNA sequence encoding Arabidopsis COI1 or ahomolog thereof may be incorporated into the host cell genome. In otherembodiments, Arabidopsis COI1 or a homolog thereof may be expressed froma vector that is not incorporated into the host cell genome. The DNAsequence encoding Arabidopsis COI1 or a homolog thereof can be placedunder the control of an endogenous promoter that is naturally present ina host cell, or it may be placed under the control of an exogenouspromoter that has been introduced into the cell in conjunction with theCOI1 or homolog sequence. In certain embodiments, Arabidopsis COI1 or ahomolog thereof may be constitutively expressed in the host cell. Inother embodiments, Arabidopsis COI1 or a homolog thereof may beexpressed in a regulated manner. In certain of these embodiments, theDNA sequence encoding COI1 or a homolog thereof may be placed under thecontrol of an inducible promoter, such as a chemically-regulatedpromoter or physically-regulated promoter. In these embodiments, theinducible promoter may provide an additional layer of control overactivation of targeted protein degradation.

In certain embodiments of the methods provided herein, Arabidopsis COI1or a homolog thereof functions in conjunction with endogenous proteinsto form a functional SCF^(CO1) E3 ligase in the non-plant cell intowhich COI1 has been introduced. For example, exogenous COI1 that hasbeen introduced into a cell may function in combination with endogenousSKP1 to form a functional E3 ligase. Since SKP1 is highly conservedamong species, the resultant complex is expected to be functional inmost non-plant cell types. Nonetheless, in certain embodiments, one ormore DNA sequences encoding SKP1 or other E3 ligase or ubiquitin pathwaycomponents may be introduced into a cell along with the one or moregenes encoding Arabidopsis COI1 or homologs thereof. These other DNAsequences may be incorporated as part of the same vector as ArabidopsisCOI1 or a homolog thereof, or they may be introduced via one or moreseparate vectors.

In certain embodiments, one or more modifications may be incorporatedinto Arabidopsis COI1 or a homolog thereof to enhance the interactionbetween COI1 or a homolog thereof and endogenous E3 ligase or otherubiquitin pathway proteins. These modifications may include one or moreadditions, substitutions, or deletions to the encoded COI1 or homologsequence. Modifications may also include the addition of one or morepeptide tags or the introduction of one or more covalent or non-covalentmodifications.

In the methods disclosed herein, a molecule that binds the COI1/JA-Ilebinding pocket of COI1 modulates the interaction between ArabidopsisCOI1 or a homolog thereof and a target protein tagged with a JAZ peptidetag. In certain embodiments, the peptide tag will only bind ArabidopsisCOI1 or a homolog thereof in the presence of the molecule that binds theCOI1/JA-Ile binding pocket of COI1. In other embodiments, the peptidetag may bind Arabidopsis COI1 or a homolog thereof with very lowaffinity in the absence of the molecule, but do so with a significantlyhigher affinity in the presence of the molecule. The addition to orremoval of the molecule that binds the COI1/JA-Ile binding pocket ofCOI1 from a cell provides a precise mechanism whereby targeted proteindegradation can be turned on and off. For example, coronatine can beintroduced into a cell to induce specific degradation, then withdrawn tohalt degradation. Thus, the methods provided herein allow for precisetemporal control of target protein degradation.

A molecule that binds the COI1/JA-Ile binding pocket of COI1 may beintroduced into a cell and/or animal via any administration pathwayknown in the art. For example, the molecule can be administered to awhole animal model via oral or parenteral administration routes.Introduction of the molecule into a host cell may be carried out via asingle administration or via multiple administrations over a set timeperiod. In certain embodiments, the molecule may be steadilyadministered to a host cell or animal over a set period of time.Withdrawal of the molecule from the host cell may occur via naturaldegradation of the molecule or by active removal or deactivation, suchas by introduction of a neutralizing molecule that degrades orinactivates the molecule.

The methods and compositions disclosed herein may be utilized fortargeted protein degradation in any non-plant host cell, including forexample eukaryotic cells such as yeast or mammalian cells. Accordingly,provided herein in certain embodiments are non-plant host cellscomprising DNA sequences encoding a target protein, a JAZ peptide tag,and Arabidopsis COI1 or a homolog thereof. Also provided herein are cellculture systems comprising such non-plant host cells.

The methods and compositions disclosed herein may also be utilized fortargeted protein degradation in whole animals and animal models.Therefore, in certain embodiments these animals and animal models arealso provided herein. In certain embodiments, the animal model is amammalian animal model, and in certain of these embodiments the mammalis a rat or mouse, such as for example a knockout mouse model.

A target protein to be tagged for degradation using the compositions andmethods disclosed herein may be an endogenous host cell protein.Alternatively, the target protein may be an exogenous protein that hasbeen stably or transiently introduced into the host cell. In thoseembodiments of the methods disclosed herein where the methods arecarried out in an animal model and wherein the target protein is anexogenous protein, DNA sequences encoding the target protein, peptidetag, and Arabidopsis COI1 or a homolog thereof may be introduced intothe animal using standard protein knockout methods known in the art. Forexample, the DNA sequences may be introduced into an embryonic stem cellunder the control of one or more exogenous or endogenous promoters. Thisstem cell may be introduced into animal blastocysts, followed byselection for progeny that are homozygous for the introduction.Alternatively, the DNA sequences may be introduced into the animal at alater stage via one or more non-plant cells comprising each of these DNAsequences or by direct transfection of one or more animal cells. Inthose embodiments wherein the methods are carried out in an animal modeland the target protein is an endogenous protein, DNA sequences encodingthe peptide tag and Arabidopsis COI1 or a homolog thereof may beintroduced into the animal by transfection of one or more animal cells.In these embodiments, the DNA sequence encoding the peptide tag isintroduced in such a manner that is it is expressed as a fusion tag tothe target protein.

In certain embodiments of the methods provided herein, targeted proteindegradation is accomplished by the steps of 1) tagging a target proteinwith one or more peptide tags comprising, consisting of, or consistingessentially of an amino acid sequence selected from the group consistingof SEQ ID NOs:5, 6, 7, and 13, b) expressing the target protein in anon-plant host cell, c) expressing Arabidopsis protein COI1 or a homologthereof in the host cell, and 4) introducing coronatine or a jasmonicacid-amino acid conjugate into the host cell, resulting in degradationof the target protein.

In certain embodiments of the methods provided herein, targeted proteindegradation in a non-plant host cell is accomplished by the steps of 1)attaching a peptide tag comprising, consisting of, or consistingessentially of an amino acid sequence selected from the group consistingof SEQ ID NOs:5, 6, 7, and 13 to a target protein, 2) introducing a DNAsequence encoding Arabidopsis COI1 or a homolog thereof into the hostcell, 3) culturing the host cell under conditions that result in theexpression of Arabidopsis COI1 or a homolog thereof, and 4) introducingcoronatine or a jasmonic acid-amino acid conjugate into the host cell,resulting in degradation of the target protein.

In certain embodiments, kits are provided for carrying out targetedprotein degradation in a non-plant host cell. In certain embodiments,these kits comprise one or more of the following components: an isolatednucleic acid encoding a JAZ peptide tag as disclosed herein, an isolatednucleic acid encoding Arabidopsis COI1 or a homolog thereof, a moleculethat binds to the coronatine/JA-Ile binding pocket of COI1, and/or aninositol pentakisphosphate cofactor. In certain embodiments, the kit mayfurther comprise a target protein or an isolated nucleic acid encoding atarget protein. In certain embodiments, these kits further compriseinstructions for usage.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention. It will be understood thatmany variations can be made in the procedures herein described whilestill remaining within the bounds of the present invention. It is theintention of the inventors that such variations are included within thescope of the invention.

EXAMPLES Example 1 Effect of Coronatine on COI1 Binding to JAZ1 and JAZ6

Various radioligand binding experiments were performed to quantify theinteraction between tritium (³H)-labeled coronatine and COI1/JAZ1 orCOI1/JAZ6.

(³H)-labeled coronatine was synthesized by Amersham. Full-lengthArabidopsis thaliana COI1 and ASK1 were co-expressed as a glutathioneS-transferase (GST) fusion protein and an untagged protein,respectively, in Hi5 suspension insect cells. The COI1/ASK1 complex wasisolated from the soluble cell lysate by glutathione affinitychromatography. After on-column tag cleavage by tobacco etch virusprotease, the complex was further purified by anion exchange and gelfiltration chromatography and concentrated by ultrafiltration to 12-18mg ml⁻¹. Full-length JAZ substrate proteins were expressed as6×His-fusion proteins in Escherichia coli and purified on Ni-NTA resinwith subsequent dialysis into 20 mM Tris-HCl, pH 8.0, 200 mM NaCl, and10% glycerol.

Radioligand binding was assayed on purified proteins, with 2 mgCOI1/ASK1 complex and JAZ proteins at a 1:3 molar ratio. Reactions wereprepared in 100 ml final volume and in a binding buffer containing 20 mMTris-HCl 200 mM NaCl, and 10% glycerol. Saturation binding experimentswere conducted with serial dilutions of 3H-coronatine in binding buffer.Nonspecific binding was determined in the presence of 300 mM coronatine.Competition binding experiments were conducted with serial dilutions ofJA-Ile in the presence of 100 nM ³H coronatine with nonspecific bindingdetermined in the presence of 300 mM coronatine. Total binding wasdetermined in the presence of vehicle only. Two-point bindingexperiments were performed in the presence of 100 nM or 300 nM³H-coronatine with nonspecific binding determined in the presence of 300mM coronatine. Following incubation with mixing at 4° C., all sampleswere collected with a cell harvester (Brandel, Gaithersburg, Md.) onpolyethyleneimine (Sigma)-treated filters. Samples were incubated inliquid scintillation fluid for >1 hour before counting with a PackardTri-Carb 2200 CA liquid scintillation analyzer (Packard Instrument Co.).Saturation binding experiments were analyzed by nonlinear regression,competition binding experiments by nonlinear regression with K_(i)calculation as described previously (Cheng 1973), andconcentration-response data by sigmoidal dose-response curve fitting,all using GraphPad Prism version 4.00 for MacOSX.

³H-coronatine showed no appreciable binding affinity for COI1,full-length JAZ1, or full-length JAZ6 alone (FIG. 1A), but bound to thecomplex of COI1/JAZ1 with a K_(D) of 48 nM and to the complex ofCOI1/JAZ6 with a K_(D) of 68 nM (FIG. 1B). Binding of coronatine to theCOI1/JAZ6 complex reached the level of saturation at 300 nM. Binding toCOI1 alone at the same concentration elicited <2% specific binding (FIG.2A). The highly active (3R,7S) isomer of JA-Ile was found to competewith coronatine for binding to the COI1-JAZ6 complex with an inhibitionconstant (K_(i)) of 1.8 μM, while the less active (3R,7R) isomercompeted with a K_(i) of 18 μM (FIG. 2B).

These results show that the COI1/JAZ complex, rather than COI1 alone,functions as the genuine high-affinity jasmonate receptor in aco-receptor form.

Example 2 Characterization of JAZ1 Peptides

The COI1-binding region of the JAZ proteins has previously been mappedto the carboxy-terminal Jas motif, which is characterized by theSLXXFXXKRXXRXXXXXPY consensus sequence preceded by two consecutive basicresidues. As shown in Example 1, mutation of the conserved phenylalanineresidue to alanine is sufficient to abolish the high affinityinteraction between coronatine and COI1/JAZ6 (FIG. 1A).

Previous studies have shown that the highly conserved PY sequence at theC-terminal end of the Jas motif plays a role in JAZ localization andstability in vivo, but that the sequence was not necessary forligand-dependent COI1-JAZ interaction. This was confirmed by anexperiment showing that truncation of the PY motif in JAZ1 had littleeffect on in vitro ligand binding activity (FIG. 3). To further map theminimal region of the Jas motif required for high affinity ligandbinding with COI1, the recombinant JAZ1 protein was replaced with a setof synthetic JAZ1 peptides in a ligand binding assay.

The JAZ1 degron of SEQ ID NO:1 (R205-Y226), which spans the centralconserved Jas motif plus the two additional amino-terminal basicresidues, did not bind to COI1 with high affinity in the presence ofcoronatine (FIG. 4). These results indicate that residues N-terminal toArg205 must participate in the COI1/JAZ interaction. Various derivativesof the JAZ1 degron sequence, as well as derivatives of the JAZ6 and JAZ7degron sequences, were analyzed for their ability to bind COI1 with highaffinity in the presence of coronatine. These derivatives are set forthin Table 1.

TABLE 1 SEQ ID Peptide NO Sequence JAZ1 Jas motif PIARRASLHRFLEKRKDRVTSKAPY JAZ1 degron  1 RRASLHRFLEKRKDRVTSKAPY JAZ1 + 1 extension  2ARRASLHRFLEKRKDRV JAZ1 + 2 extension  3 IARRASLHRFLEKRKDRV JAZ1 +3 extension  4 PIARRASLHRFLEKRKDRV JAZ1 + 4 extension  5LPIARRASLHRFLEKRKDRV JAZ1 + 5 extension  6 ELPIARRASLHRFLEKRKDRV JAZ1 +6 extension +  7 ELPIARRASLHRFLEKRKDRVT PY motif SKAPY JAZ1 + polyA  8AAAAARRASLHRFLEKRKDRV extension JAZ1 + 5 extension  9VERIARRASLHRFLEKRKDRV from JAZ6 JAZ6 + 5 extension 10VERIARRASLHRFFAKRKDRV JAZ6 + 10 11 QQHQVVERIARRASLHRFFAKR extension KDRVJAZ7 + 5 extension 12 YQKASMKRSLHSFLQKRSLRI

The JAZ1 +1, +2, +3, +4, and +5 extensions (SEQ ID NOs:2-6,respectively) added on 1, 2, 3, 4, and 5 residues, respectively, to theN-terminus of JAZ1 while removing six residues from the C-terminus. Theadded residues were derived from the residues that are normally presentN-terminal to the degron in JAZ1. An additional JAZ1 peptide (SEQ IDNO:7) contained the same additional N-terminal amino acids as the +5extension but with the six C-terminal residues added back on.

The JAZ1 +4 and +5 extensions (SEQ ID NOs:5 and 6, respectively) werefound to bind COI1 in the presence of coronatine with a much higheraffinity than the JAZ1 degron and the +1, +2, and +3 extensions, withthe +5 extension exhibiting the highest degree of binding (FIG. 4). TheJAZ1 +5 extension peptide was found to permit coronatine binding with aK_(D) (˜108 nM) comparable to that of the full-length JAZ1 protein (FIG.5). The JAZ1 +5 extension with the six C-terminal residues added back onalso exhibited significant binding (FIG. 6, “JAZ1-extension +5+PY”).

To test the specificity of the JAZ1 peptide tags of SEQ ID NOs:5, 6, and7, an additional set of JAZ peptide tags was developed. The first twowere essentially identical to the JAZ1 +5 extension in that theycontained residues 1-16 of the JAZ1 degron plus a five amino acidN-terminal extension. However, the sequence of the extension wasdifferent. The first of these (SEQ ID NO:8) utilized a polyalanineextension, while the second (SEQ ID NO:9) utilized a five amino acidextension derived from JAZ6. The remaining JAZ peptide tags were basedon the degrons of JAZ6 and JAZ7. These included two JAZ6 peptides thatcontained the JAZ6 degron plus an additional five and ten N-terminalresidues, respectively (SEQ ID NOs: 10 and 11, respectively) and oneJAZ7 peptide that contained the JAZ7 degron plus an additional fiveN-terminal residues (SEQ ID NO: 12). None of the additional JAZ peptidetags exhibited significant binding to COI1 in the presence of coronatine(FIGS. 4 and 6). These results suggest that the system disclosed hereinmaintains a great deal of selectivity for side chain chemistry, and thatsimply inserting five “filler” amino acid residues at the N-terminus ofthe peptide tag is insufficient to promote COI1 binding.

Example 3 Structural Relationship of COI1/JAZ1 and Coronatine

To evaluate the structure mechanism by which COI1/JAZ1 co-receptorsenses jasmonate, crystal structures were obtained for COI1/ASK1/JAZ1peptides complexed with either coronatine or (3R,7S)-JA-Ile.

Crystals were grown at 4° C. by the hanging-drop vapor diffusion methodwith 1.5 μL protein complex samples containing COI1/ASK1, JAZ1 peptide,and hormone compound at 1:1:1 molar ratio mixed with an equal volume ofreservoir solution containing 100 mM BTP, 1.7-1.9 M ammonium phosphate,and 100 mM NaCl, pH 7.0. Diffraction quality crystals were obtained bythe micro-seeding method at 4° C. The crystals all contained eightcopies of the complex in the asymmetric unit. The data sets werecollected at the Advanced Light Source in Lawrence Berkeley NationalLaboratory as well as the GM/CA-CAT 23 ID-B beamline at the AdvancedPhoton Source in Argonne National Laboratory using crystals flash-frozenin the crystallization buffers supplemented with 15-20% ethylene glycolat −170° C. Reflection data were indexed, integrated, and scaled withthe HKL2000 package. All crystal structures were solved by molecularreplacement using the program Phaser and the TIR1/ASK1 structure assearch model. The structural models were manually built in the program Oand refined using CNS and PHENIX. All final models had 96-98% ofresidues in the favored region and 0% in disallowed region of theRamachandran plot.

TABLE 2 COI1/ASK1/JAZ1 COI1/ASK1/JAZ1 +5 extension/ COI1/ASK1/JAZ1degron/coronatine coronatine +5 extension/JA-Ile Data collection Spacegroup P21 P21 P21 Cell a, b, c 121.8, 221.5, 148.5 123.2, 220.8, 149.5122.3, 220.8, 148.7 dimensions (Å) α, β, γ 90.0, 104.5, 90.0 90.0,104.5, 90.0 90.0, 104.5, 90.0 (°) Resolution (Å) 2.80 (2.80-2.90) 3.35(3.35-3.41) 3.18 (3.18-3.31) R_(sym) 0.103 (0.816) 0.119 (0.700) 0.088(0.462) //σ/ 16.7 (2.0) 14.0 (2.1) 17.2 (2.8) Completeness (%) 100 (100)92.9 (94.6) 97.0 (93.3) Redundancy 3.9 (3.8) 3.6 (3.3) 3.1 (2.7)Refinement Resolution (Å) 50-2.80 50-3.35 50-3.18 No. reflections174,966 95,997 116,337 R_(work)/R_(free) 0.235/0.270 0.225/0.2700.223/0.264 R.m.s Bond 0.008 0.010 0.010 deviations lengths (Å) Bond1.676 1.271 1.556 angles (°)

The crystal structure of COI1 revealed a TIR1-like overall architecture,with the N-terminal tri-helical F-box motif bound to ASK1 and aC-terminal horseshoe-shaped solenoid domain formed by 18 tandemleucine-rich repeats (FIGS. 7A and 7B). Similar to TIR1, the top surfaceof the COI1 leucine-rich repeat (LRR) domain has three long intra-repeatloops (loops 2, 12, and 14) that are involved in hormone and polypeptidesubstrate binding. Unlike TIR1, however, a fourth long loop (loop C) inthe C-terminal capping sequence of the COI1 LRR domain folds over loop2, partially covering it from above (FIGS. 7B and 7C).

Despite their similar overall fold, crystal structure analysis revealedthat COI1 has evolved a hormone binding site that is distinct from thatof TIR1. Configured between loop 2 and the inner wall of the LRR, theligand binding pocket of COI1 is exclusively encircled by amino acidside chains (FIG. 8). Many of the pocket-forming residues on COI1 arelarge in size and carry a polar head group (FIG. 9). These propertiesallow them to be mold a binding pocket into a specific shape whileforming close interactions with each chemical moiety of the ligand.These close interactions are critical to proper hormone sensing of thecomplex. In the binding pocket, both JA-Ile and coronatine sit in an‘upright’ position with the keto group of their common cyclopentanonering pointing up and forming a triangular hydrogen bond network withR496 and Y444 of COI1 at the pocket entrance (FIG. 8).

Without the JAZ degron peptide bound, the keto group of the ligand isaccessible to solvent (FIG. 10). The rest of the cyclopentanone ring ofboth JA-Ile and coronatine is sandwiched between the aromatic groups ofF89 and Y444 of COI1, stabilized by hydrophobic packing. The cyclohexenering of coronatine provides a rigid surface area for close packing withF89, whereas the more flexible and extended pentenyl side chain ofJA-Ile is more loosely accommodated by a hydrophobic pocket formed byA86, F89 and L91 from loop 2 as well as L469 and W519 from the LRRs(FIG. 11A). Differences at this interface probably explain theapproximately tenfold higher affinity of coronatine over (3R,7S)-JA-Iledetected in binding assays. Deeper in the ligand-binding pocket, thecommon amide and carboxyl groups of JA-Ile and coronatine bind to thebottom of the binding site by forming a salt bridge and hydrogen bondnetwork with three basic residues of COI1:R85, R348 and R409 (FIGS. 8Aand 8B). Together, these arginine residues constitute the charged floorof the ligand pocket. Y386 reinforces the interactions from above byforming a hydrogen bond with the amine group of the ligand. In doing so,Y386 approaches the cyclopentanone ring of the ligand, narrowing thepocket entrance and creating a hydrophobic cave below. The rest of thebasin is carved out by V411, A384 and the aliphatic side chain of Arg409 (FIG. 11B). The ethyl-cyclopropane group of coronatine and theisoleucine side chain of JA-Ile can both comfortably fit in this spacedue to their similar size and hydrophobicity. The nature of the caveexplains the preference of COI1 for jasmonate conjugates containing amoderately sized hydrophobic amino acid. Although most of the ligand isburied inside the binding site, the keto group at the top and thecarboxyl group at the bottom remain exposed, available for additionalinteractions with the JAZ portion of the co-receptor (FIG. 10).

The JAZ1 degron peptide adopts a bipartite structure with a loop regionfollowed by an α-helix to assemble with the COI1/JA complex. Thehallmark of the JAZ1 degron is the N-terminal five amino acidsidentified in the radioligand binding assay. In a largely extendedconformation, this short sequence lies on top of the hormone-bindingpocket and simultaneously interacts with both COI1 and the ligand,effectively trapping the ligand in the pocket (FIGS. 12A and 12B). Atthe N-terminal end, L201 of the JAZ1 peptide is embedded in ahydrophobic cavity presented by surface loops on top of COI1 (FIG. 13A).

At the C-terminal end, A204 of JAZ1 uses its short side chain to packagainst the keto group of the ligand and F89 of COI1 (FIGS. 11A and13A). The same alanine residue of JAZ1 also donates a hydrogen bondthrough its backbone amide group to the keto moiety of the ligandemerging from the pocket (FIG. 13A). The middle region of thefive-amino-acid sequence is secured to the COI1 jasmonate complexthrough a hydrogen bond formed between the backbone carbonyl of P202 inJAZ1 and the ligand-interacting COI1 residue R496. which is critical forthe hormone-dependent COI1/JAZ interaction (data not shown). Inagreement with its important role in forming the JA-Ile co-receptor,this short N-terminal region of the JAZ degron completely covers theopening of the ligand-binding pocket, conferring high-affinity bindingto the hormone. The close interaction between the hormone and theco-receptor complex provides a plausible structural explanation for thefavorable binding of the (3R,7S)-JA-Ile isomer, as the stereochemistryat the 7 position of (3R,7R)-JA-Ile may place the aliphatic chainunfavorably close to nearby JAZ1 and COI1 residues (FIG. 11A).

Within the JAZ1 degron, two conserved basic residues, R205 and R206,were previously shown to have an important role in hormone-induced COI1binding. In the structure, R205 contributes to COI1 binding by directlyinteracting with loop 12, whereas R206 points in the opposite directionand inserts deeply into the central tunnel of the COI1 solenoid.Approaching the bottom of the ligand-binding pocket, the guanidiniumgroup of the R206 side chain joins the three basic COI1 residues thatform the pocket floor and interacts directly with the carboxyl group ofthe ligand (FIG. 13B). Thus, the N-terminal seven amino acids (ELPIARR)of the JAZ1 degron peptide act as a clamp that wraps the ligand-bindingpocket from top to bottom, closing it completely (FIG. 12B).

The highly conserved C-terminal half of the JAZ1 degron forms anamphipathic α-helix that strengthens the JAZ1/COI1 interaction bybinding to the top surface of the COI1 LRR domain, adjacent to theligand-binding site (FIG. 12A). With its N-terminal end directly packingagainst loop 2 of COI1, the Jas motif helix blocks the central tunnel ofthe COI1 LRR solenoid like a plug. The N-terminal half of the Jas motifhelix is characterized by three hydrophobic residues (L209, F212 andL213) which are aligned on the same side of the helix and form ahydrophobic interface with COI1 (FIG. 13C).

By soaking the COI1-ASK1 crystals with coronatine and a sufficientlyhigh concentration of JAZ1 degron peptide lacking the N-terminal ELPIAsequence, the complex formed by COI1, coronatine, and the isolated Jasmotif helix was trapped in the crystal (Table 2). This indicates thatthe α-helix may provide a low-affinity anchor for docking the JAZprotein on COI1.

Example 4 Identification of COI1 Cofactor

The crystal structure of TIR1 revealed an unexpected inositolhexakisphosphate (InsP₆) molecule bound in the centre of the proteinunderneath the auxin-binding pocket. Sequence homology between COI1 andTIR1 suggests that COI1 might contain a similar small molecule. Beforecrystallization, the recombinant COI1/ASK1 complex was analyzed bystructural mass spectrometry.

Nano-electrospray ionization mass spectrometry (MS) and tandem MS(MS/MS) experiments were performed on a SynaptHDMS instrument. Before MSanalysis, 50 μL of a 16 mg ml⁻¹ solution of COI1/ASK1 in 20 mM Tris-HCl,pH 8, 0.2 M NaCl, and 5 mMDTT was buffer-exchanged twice into 0.5Mammonium acetate solution using Bio-Rad Biospin columns. To improvedesolvation during ionization, samples were diluted 1:4 in 0.5 Mammonium acetate, and isopropanol was added to a final concentration of5%. Typically an aliquot of 2 mL solution was loaded for sampling vianano-ESI capillaries, which were prepared in-house from borosilicateglass tubes as described previously (Nettleton 1998). The conditionswithin the mass spectrometer were adjusted to preserve non-covalentinteractions. The following experimental parameters were used: capillaryvoltage up to 1.26 kV, sampling cone voltage 150 V, and extraction conevoltage 6 V, MCP 1590. For tandem MS experiments peaks centered at m/z4,564 and 4,588 were selected in the quadrupole and collision energy upto 65 V was used. Argon was used as a collision gas at maximum pressure.All spectra were calibrated externally using a solution of cesium iodide(100 mg ml⁻¹). Spectra are shown with minimal smoothing and withoutbackground subtraction.

Nano-electrospray mass spectra of the intact COI1/ASK1 complex revealedtwo populations differing by a mass of ˜568 Da, indicating that a smallmolecule was indeed co-purified with the proteins (FIGS. 15 and 16). Asshown in FIG. 16, only one population of the complex corresponding toCOI/ASK1 was apparent at both at low and high collision energy. At highcollision energy, the COI/ASK1 complex dissociates into its differentsubunits and one population of COI1 appears in the spectrum. Thispopulation of COI1 has a calculated mass of 67,944±1 Da, which is inagreement with the theoretical mass of COI1 (67,947 Da). At lowcollision energy, the COI1/ASK1/ligand complex is apparent. However,elevating the collision energy releases some of the bound ligand andresults in the appearance of a stripped COI1/ASK1 complex. Thetheoretical mass of the apo COI1/ASK1 complex is 86,458 Da, which is inclose agreement with the observed mass of 86,543±28 Da. The mass of theCOI1/ASK1/ligand complex was found to be 87,112±15 Da, suggesting thatthe mass of the ligand is around 568±28 Da. The fact that both massescarry a charge of +19 indicates a neutral loss of the ligand, meaningthat it cannot be detected in the spectrum. At high collision energy,some of the complex dissociates into its different subunits and twopopulations of COI1 appear in the spectrum. The smaller form, with acalculated mass of 67,952±5 Da, fits the theoretical mass of COI1(67,946.5 Da), whereas the other population, with a calculated mass of68,518±4 Da, corresponding to COI1-ligand, suggest that the mass of theligand is around 568±5 Da.

The mass-spectrometry-derived molecular mass of the unknown compound isdifferent from the mass of nsP₆ (651 Da) but matches that of an inositolpentakisphosphate (InsP₅) molecule. Unfortunately, mass spectrometryanalyses of either the native COI1/ASK1 complex or the denaturedproteins were unable to achieve direct mass analysis of the smallmolecule.

To investigate the identity of the unknown compound, it was firstestimated that the molecule contains four or five phosphate groups by³¹P nuclear magnetic resonance (NMR) of trypsin-digested COI1/ASK1complex (data not shown). To identify unequivocally the unknownmolecule, steps were taken to purify it away from the COI1/ASK1 complexin a quantity sufficient for ¹H NMR analysis. The high phosphate contentof the molecule allowed us to trace it through a multi-step purificationprocedure (FIG. 17). Phenol was melted at 68° C. and equilibrated withequal parts 0.5 M Tris-HCl, pH 8.0 until a pH of 7.8 was reached. Theequilibrated phenol was then topped with 0.1 volume 100 mM Tris-HCl, pH8.0 and stored at 4° C. For extraction, 30-40 mg of 1 mg ml⁻¹ COI1/ASK1protein was mixed in small batches with equal parts equilibrated phenolat room temperature. The samples were inverted and incubated for 30minutes until phase separation occurred. With 30 second vortexing, thesamples were incubated at room temperature for 30 minutes and spun at15,000 rpm for 5 minutes. The aqueous phase was removed as a primaryextraction. Equal parts of a solution containing 25 mM Tris-HCl, pH 8.0was added to the phenol and collected as above as a secondaryextraction. The primary and secondary extractions were combined anddiluted 10× in 25 mM Tris-HCl, pH 8.0, then further purified by gravityflow on Q sepharose high-performance anion exchange resin (GEHealthcare). Following column wash with 10× column volumes of 0.1 Nformic acid, stepwise elution was performed with 23 column volumes of0.1 N formic acid (Thermo Scientific) with increasing concentrations ofammonium formate (Sigma) from 0 to 2 M. Fractions were analyzed forphosphate content by the wet-ashing method with perchloric acid in Pyrexculture tubes (13×100 mm). Typically, samples of 50-100 μL were ashedwith 100-200 mL 70% perchloric acid (purified by redistillation, Sigma).Ashing was performed by heating the sample over a Bunsen-type burnerwith continuous shaking to prevent bumping. When the sample stoppedemitting white smoke, the reaction was considered complete and thenheated to dryness. 500 μL of distilled water was added to the roomtemperature tubes and vortexed. 100 μL samples containing up to 10 nmolinorganic phosphate were assayed for phosphate by a modification of apublished procedure (Sadrzadeh 1993). A total of 125 μL of acidmolybdate color reagent was added and the samples were incubated andcovered at room temperature for 12-14 hours (overnight) for full colordevelopment (total volume 225 μL). Plates were read at 650 nm andunknowns were determined from the linear regression of the standardcurve (0-10 nmol NaH₂PO₄ per well). All assays were done in triplicate.Final fractions containing phosphate were combined and lyophilizedrepeatedly to remove residual ammonium formate.

After isolation of 150 nmol of the purified small molecule, a series ofone-dimensional and two-dimensional NMR data were acquired, including ahighly informative homonuclear total correlation (TOCSY) spectrum. NMRspectra were acquired on a Varian INOVA600 spectrometer equipped with acold probe using 200 μM samples of purified compound X or syntheticinositol-1,2,4,5,6-pentakisphosphate (Cayman Chemical) dissolved in D₂O.TOCSY spectra were acquired with mixing times of 35 or 50 ms, processedwith NMRPipe and visualized with NMRView.

The observed chemical shifts and TOCSY cross-peak patterns are clearlycharacteristic of inositol phosphates (FIG. 18). A comparison withpreviously reported NMR spectra of various inositol phosphatesestablished that the unknown compound is either D- orL-inositol-1,2,4,5,6-pentakisphosphate (Ins(1,2,4,5,6)P₅; FIG. 18). Thisconclusion was further supported by the TOCSY spectrum of syntheticIns(1,2,4,5,6)P₅ (FIG. 19) and the subsequently acquired negative ionelectrospray ionization mass spectrometry spectrum of the compound (FIG.20).

As shown in FIG. 20, the negative-ion ESI-MS spectrum of the unknowncontained the major ion at m/z 192.3 ((579.8951−3×1.0078)/3),corresponding to the [M-3H]³⁻ ion of inositol pentakisphosphate (InsP₅),and the ion at m/z 288.9 ((579.8951−2×1.0078)/2), corresponding to the[M-2H]²⁻ ion of InsP₅. The [M-H]⁻ ion expected at m/z 579.9 was absent.The ions seen at m/z 199.7 and 207.1 correspond to the sodiated ions ofInsP₅ seen as the [M+Na-4H]³⁻, and [M+2Na-5H]³ ions, respectively; andthe ions at m/z 299.9 and 311.9 correspond to the [M+Na-3H]²⁻ and[M+2Na-4H]²⁻ ions, respectively. The spectrum also contains ions at m/z499 ([M-H-HPO₃]⁻), 419 ([M-H-2HPO₃]⁻), and 441 ([M+Na-2H-2HPO₃]⁻),arising from various losses of the phosphate residues of the molecule.The presence of the ion at m/z 499 (579.9-HPO₃) is consistent with theobservation of the ions at m/z 249 ([M-2H-HPO₃]⁻²), 259.9([M+Na-3H-HPO₃]⁻²), and 165.7 ([M-3H-HPO₃]⁻³), representing the variousdeprotonated InsP₄ seen as doubly and triply charged anions. The ion atm/z 419 represents a deprotonated InsP₃ arising from loss of two HPO₃residues; while the ion at m/z 441 represents a monosodiated InsP₃anion. The presence of the ions at m/z 419 and 441 is also consistentwith the observation of the doubly charged ions at m/z 209 and 219,corresponding to the [M-2H-2HPO₃]⁻² and [M+Na-3H-2HPO₃]⁻² ions,respectively. The assignments of the ions observed are listed in Table3. These ions were also observed for Ins(1,2,3,4,5)P₅ andIns(1,2,4,5,6)P₅ standards when subjected to ESI under the samecondition, indicating that the unknown compound is an InsP₅. This InsP₅structure is further confirmed by the MSn (n=2,3,4,5) mass spectra ofthe [M-3H]³⁻ ion at m/z 192.3 and of the [M-2H]²⁻ ion at m/z 288.9deriving from the unknown compound and from the Ins(1,2,3,4,5)P₅ andIns(1,2,4,5,6)P₅ standards.

TABLE 3 Ions observed for IP5 by negative-ion ESI-MS m/z Structure 499[M − H − HPO₃]⁻ 441 [M + Na − 2H − 2HPO₃]⁻ 419 [M − H − 2HPO₃]⁻ 311 [M +2Na − 4H]⁻² 300 [M + Na − 3H]⁻² 289 [M − 2H]⁻² 271 [M + 2Na − 2H −2HPO₃]⁻² 268 [M + K − 3H − 2HPO₃]⁻² 259.9 [M + Na − 3H − HPO₃]⁻² 249 [M− 2H − HPO₃]⁻² 219 [M + Na − 3H − 2HPO₃]⁻² 212 [M + Na + K − 5H]⁻³ 209[M − 2H − 2HPO₃]⁻² 207 [M + 2Na − 5H]⁻³ 203 [M + Na − 4H]⁻³ 199.7 [M +Na − 4H]⁻³ 192.3 [M − 3H]⁻³ 165.7 [M − 3H − HPO₃]⁻³ 97 H₂PO₄ ⁻ 79 PO₃ ⁻

Consistent with the binding of a small molecule cofactor, the crystalstructure of COI1 (Example 3) showed strong unexplained electrondensities clustered in the middle of the COI1 LRR domain. Like InsP₆ inTIR1, these extra densities in COI1 are located directly adjacent to thebottom of the ligand binding pocket of the jasmonate co-receptor,interacting with multiple positively charged COI1 residues (FIG. 21A).Unexpectedly, these islands of electron density cannot be explained byan Ins(1,2,4,5,6)P₅ molecule. Instead, their intensity, overallsymmetry, and poor connectivity indicate that they belong to multiplefree phosphate molecules. Because a high concentration of ammoniumphosphate was used as the major precipitant for crystallizing the JAco-receptor, it was postulated that the InsP₅ molecule that co-purifiedwith COI1 was later displaced by phosphate molecules in thecrystallization drops. In support of this scenario, the concave surfaceof the COI1 solenoid fold surrounding the phosphates is highly basic anddecorated with residues conserved in plant COI1 orthologs, indicating afunctionally important surface area (FIGS. 9, 21B, 22).

The highly selective co-purification of two different inositolphosphates, InsP₅ and InsP₅, with two homologous plant hormonereceptors, COI1 and TIR1, implies that the proper function of the twoF-box proteins might require the binding of specific inositolphosphates. To assess the functional role of Ins(1,2,4,5,6)P₅ in theCOI1/JAZ1 co-receptor, a protocol was developed for stripping theco-purified InsP₅ from COI1 without denaturing the protein. Briefly,proteins were mixed with 10% glycerol and incubated in 2 M ammoniumphosphate, 100 mM Bis-Tris propane, pH 7.0, 200 mM NaCl, and 10%glycerol at 4° C. for >24 hours with a minimum of 3× buffer changes at100× sample volume. Samples were then transferred to 20 mM Tris-HCl, pH8.0, 200 mM NaCl, and 10% glycerol at 4° C. for >24 hours with a minimumof three buffer changes at 100× sample volume. Inositol phosphate rescueexperiments were conducted according to the radioligand binding assaysdescribed above in the presence of 300 nM ³H-coronatine with nonspecificbinding determined in the presence of 300 μM coronatine.

The resulting COI1/ASK1 complex was tested in a ligand-binding basedreconstitution assay. As shown in FIG. 23A, untreated COI1 formed ahigh-affinity jasmonate co-receptor with JAZ1. Addition of exogenousIns(1,2,4,5,6)P₅ did not significantly change its activity. In contrast,the dialyzed COI1 sample completely lacked ligand binding by itself andshowed only trace activity in the presence of JAZ1. Supplementation witheither synthetic Ins(1,2,4,5,6)P₅ (FIG. 23B) or the purified and NMRanalyzed InsP₅ sample (data not shown) rescued the interaction in adose-dependent manner and with a half-maximum effective concentration(EC₅₀) of 27 nM (FIG. 23C). From this reconstitution result, it wasconcluded that Ins(1,2,4,5,6)P₅ binding is crucial for the jasmonatecoreceptor to perceive the hormone with high sensitivity.

A close examination of the phosphate molecules in the available COI1structure indicates a mechanism by which the inositol phosphate moleculemay modulate the activity of the jasmonate co-receptor. Among fourCOI1-bound phosphates, one stands out by binding at a critical positionin the jasmonate co-receptor. This phosphate molecule interactssimultaneously with four basic residues at the bottom of theligand-binding pocket, namely Arg 206 in the JAZ1 degron and the threeCOI1 arginine residues that form the floor of the pocket. As a result, atetragonal bipyramidal interaction network is formed among fourmolecules at the core of the jasmonate co-receptor assembly. The fourarginines from COI1 and JAZ1 sit at the four corners of the centralplane, interacting with the hormone above and the phosphate below (FIG.24).

As the free phosphate molecule probably mimics the action of a phosphategroup on InsP₅, this four-molecule junction, together with additionalphosphate-COI1 interactions seen in the crystal, conceivably representsthe structural basis for InsP₅ potentiation of the jasmonate coreceptor.Consistent with this interpretation, coronatine-induced formation of aCOI1/JAZ1 complex was readily abolished by mutation of select COI1residues adjacent to the phosphates, but not in contact with the hormone(FIG. 25).

The reconstitution assay was used to further investigate the specificityof jasmonate co-receptor regulation by inositol phosphates (FIG. 14A).Notably, inositol-1,4,5,6-tetrakisphosphate supports the activity of theCOI1/JAZ1 co-receptor, whereas the second messenger signaling moleculeinositol-1,4,5-trisphosphate does not. Addition of a phosphate to InsP₅,which gives rise to InsP₆, is also not favorable for activity. Althoughsaturation binding of ³H-coronatine is stimulated by bothIns(1,2,4,5,6)P₅ and InsP₆ with similar 1K_(d) values (30 nM and 37 nM,respectively), the two inositol phosphates yield markedly differentB_(max) values for coronatine binding, indicating that InsP₆ issignificantly less efficacious in activating the co-receptor despitehaving equal affinity as Ins(1,2,4,5,6)P₅ (FIG. 14B). Functionalselectivity of COI1 for the inositol phosphate cofactor is consistentwith the conservation of the putative inositol-phosphate-binding site,which is distinct in amino acid sequence from the InsP₆-binding site inTIR120 (FIG. 9).

Example 5 Targeted Degradation of a Target Protein

Green fluorescent protein (GFP) will be tagged with the JAZ1 +5extension peptide tag of SEQ ID NO:6 in budding yeast cells (e.g.,Saccharomyces cerevisiae) and/or mammalian cells. Where budding yeastcells are used, the tagged protein construct will be cloned into astandard yeast shuttling plasmid under the control of a strong, stablepromoter, and the plasmid will be stably inserted into the yeast genomevia chromosomal recombination sequences using methods well known in theart. Where mammalian cells are used, the gene encoding the taggedprotein construct will be introduced via transient transfection orstable cell line generation.

The cells will be further engineered to express Arabidopsis COI1 or ahomolog thereof under the control of an inducible promoter. For example,exogenous COI1 expression may be placed under the control of a galactosepromoter, such that expression may be controlled by sugar ratio.

After stable GFP signal has been monitored qualitatively using standardtechniques such as microscopy and/or quantitatively using techniquessuch as standard plate readers and/or flow cytometry methods, expressionof Arabidopsis COI1 or a homolog thereof will be induced. COI1expression should not significantly affect GFP signal levels.

Cells will be treated with titrating levels of coronatine. Cells will beharvested and fixed at various timepoints, and GFP signal will bequantified to determine the rate of GFP degradation. Degradation willincrease as coronatine levels increase.

Additional experiments may be performed using one or more of the otherJAZ1 peptide tags disclosed herein to determine the efficacy of slightchanges to the peptide sequence. Similarly, additional experiments maybe performed using molecules other than coronatine that bind to theCOI1/JA-Ile binding pocket of COI1.

As stated above, the foregoing is merely intended to illustrate variousembodiments of the present invention. The specific modificationsdiscussed above are not to be construed as limitations on the scope ofthe invention. It will be apparent to one skilled in the art thatvarious equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein. All referencescited herein are incorporated by reference as if fully set forth herein.

REFERENCES

-   1. Adams Acta Crystallogr D 58:1948-1954 (2002)-   2. Browse Annu Rev Plant Biol 60:183-205 (2009)-   3. BrUnger Acta Crystallogr D 54:905-921 (1998)-   4. Cheng Biochem Pharmacol 22:3099-3108 (1973)-   5. Chini Nature 448:666-671 (2007)-   6. Chung Plant Cell 21:131-145 (2009)-   7. Chung Plant J 63:613-622 (2010)-   8. Delaglio J Biomol NMR 6:277-293 (1995)-   9. Dharmasiri Nature 435:441-445 (2005)-   10. Feys Plant Cell 6:751-759 (1994)-   11. Fonseca Nature Chem Biol 5:344-350 (2009)-   12. Grunewald EMBO Rep 10:923-928 (2009)-   13. Johnson Methods Mol Biol 278:313-352 (2004)-   14. Jones Acta Crystallogr A 47:110-119 (1991)-   15. Katsir Proc Natl Acad Sci USA 105:7100-7105 (2008)-   16. Kepinski Nature 435:446-451 (2005)-   17. Koo Plant J 59:974-986 (2009)-   18. Lorenzo Plant Cell 16:1938-1950 (2004)-   19. Melcher Nature 462:602-608 (2009)-   20. Melotto Plant J 55:979-988 (2008)-   21. Miyazono Nature 462:609-614 (2009)-   22. Murase Nature 456:459-463 (2008)-   23. Nettleton J Mol Biol 281:553-564 (1998)-   24. Nishimura Nature Methods 6:917-922 (2009)-   25. Nishimura Science 326:1373-1379 (2009)-   26. Ogawa Tetrahedr Lett 49:7124-7127 (2008)-   27. Sadrzadeh J Pharmacol Toxicol Methods 30:103-110 (1993)-   28. Sakamoto Proc Natl Acad Sci USA 98:8554-8559 (2000)-   29. Santiago Nature 462:665-668 (2009)-   30. Sheard Nature 468:400-405 (2010)-   31. Shimada Nature 456:520-523 (2008)-   32. Staswick Plant Cell 16:2117-2127 (2004)-   33. Stephens Biochem J 275:485-499 (1991)-   34. Suza Planta 227:1221-1232 (2008)-   35. Tan Nature 446:640-645 (2007)-   36. Thines Nature 448:661-665 (2007)-   37. Xie Science 280:1091-1094 (1998)-   38. Yan Plant Cell 19:2470-2483 (2007)-   39. Yan Plant Cell 21:2220-2236 (2009)-   40. Yin Nature Struct Mol Biol 16:1230-1236 (2009)-   41. Zhang Proc Natl Acad Sci USA 100:14127-14132 (2003)-   42. Zhou Mol Cell 6:751-756 (2000)

1. A method for targeted protein degradation comprising: a) tagging atarget protein with one or more peptide tags, wherein said peptide tagscomprise an amino acid sequence selected from the group consisting ofSEQ ID NOs:5, 6, 7, and 13; b) expressing said target protein in anon-plant host cell; c) expressing Arabidopsis protein COI1 or a homologthereof in said host cell; and d) introducing a molecule that binds theCOI1/JA-Ile binding pocket of COI1 into said host cell, whereinintroduction of a molecule that binds the COI1/JA-Ile binding pocket ofCOI1 results in degradation of said target protein.
 2. The method ofclaim 1, wherein said non-plant host cell is selected from the groupconsisting of a yeast and a mammalian cell.
 3. The method of claim 1,wherein said Arabidopsis protein COI1 or homolog thereof is selectedfrom the group consisting of Arabidopsis thaliana COI1 (SEQ ID NO:15),Arabidopsis lyrata COI1 (SEQ ID NO:16), Oryza sativa COI1 (SEQ ID NO:17), Solanum lycopersicum COI1 (SEQ ID NO: 18), Vitis vinifera COI1 (SEQID NO: 19), Populus trichocarpa COI1 (SEQ ID NO:20 and/or SEQ ID NO:21),Ricinis communis COI1 (SEQ ID NO:22), Zea mays COI1 (SEQ ID NO:23),Hevea brasiliensis COI1 (SEQ ID NO:24), Pisum sativum COI1 (SEQ IDNO:25), Nicotiana attenuate COI1 (SEQ ID NO:26), Glycine max COI1 (SEQID NO:27), Sorghum bicolor COI1 (SEQ ID NO:28), wheat COI1, andPhyscomitrella patens COI1.
 4. The method of claim 1, wherein saidtarget protein is endogenous to said non-plant host cell.
 5. The methodof claim 1, wherein said target protein is exogenous to said non-planthost cell.
 6. The method of claim 1, wherein said molecule that bindsthe COI1/JA-Ile binding pocket of COI1 is coronatine or a jasmonicacid-amino acid conjugate.
 7. The method of claim 6, wherein saidjasmonic acid-amino acid conjugate is JA-Ile.
 8. The method of claim 1,further comprising the step of introducing an inositol pentakisphosphatecofactor into said host cell.
 9. A method for targeted proteindegradation in a non-plant host cell comprising: a) attaching a peptidetag to a target protein, wherein said peptide tag consists essentiallyof an amino acid sequence selected from the group consisting of SEQ IDNOs:5, 6, 7, and 13; b) introducing a first DNA sequence encodingArabidopsis COI1 or a homolog thereof into said host cell; c) culturingsaid non-plant host cell under conditions that result in expression ofArabidopsis COI1 or a homolog thereof and said target protein attachedto said peptide tag; and d) introducing a molecule that binds theCOI1/JA-Ile binding pocket of COI1 into said host cell, whereinintroduction of said molecule that binds the COI1/JA-Ile binding pocketof COI1 results in degradation of said target protein attached to saidpeptide tag.
 10. The method of claim 9, wherein said non-plant host cellis selected from the group consisting of a yeast and a mammalian cell.11. The method of claim 9, wherein said Arabidopsis COI1 or a homologthereof is selected from the group consisting of Arabidopsis thalianaCOI1 (SEQ ID NO:15), Arabidopsis lyrata COI1 (SEQ ID NO:16), Oryzasativa COI1 (SEQ ID NO:17), Solanum lycopersicum COI1 (SEQ ID NO:18),Vitis vinifera COI1 (SEQ ID NO:19), Populus trichocarpa COI1 (SEQ IDNO:20 and/or SEQ ID NO:21), Ricinis communis COI1 (SEQ ID NO:22), Zeamays COI1 (SEQ ID NO:23), Hevea brasiliensis COI1 (SEQ ID NO:24), Pisumsativum COI1 (SEQ ID NO:25), Nicotiana attenuate COI1 (SEQ ID NO:26),Glycine max COI1 (SEQ ID NO:27), Sorghum bicolor COI1 (SEQ ID NO:28),wheat COI1, and Physcomitrella patens COI1.
 12. The method of claim 9,wherein said molecule that binds the COI1/JA-Ile binding pocket of COI1is coronatine or a jasmonic acid-amino acid conjugate.
 13. The method ofclaim 12, wherein said jasmonic acid-amino acid conjugate is JA-Ile. 14.The method of claim 9, further comprising the step of introducing aninositol pentakisphosphate cofactor into said host cell.
 15. The methodof claim 9, wherein said target protein is endogenous to said non-planthost cell, and wherein step (a) comprises introducing a second DNAsequence encoding said peptide tag into said host cell such that saidsecond DNA sequence is incorporated adjacent to a third DNA sequenceencoding said target protein, wherein said peptide tag is expressed asan attachment to said target protein.
 16. The method of claim 9, whereinsaid target protein is exogenous to said non-plant host cell, andwherein step (a) comprises introducing a second DNA sequence encodingsaid target protein and said peptide tag, wherein said peptide tag isexpressed as an attachment to said target protein.
 17. A method fortargeted protein degradation in a host animal comprising: a) introducinga first DNA sequence encoding a target protein linked to a peptide tagand a second DNA sequence encoding Arabidopsis COI1 or a homolog thereofinto one or more cells of said animal, wherein said peptide tag consistsessentially of an amino acid sequence selected from the group consistingof SEQ ID NOs:5, 6, 7, and 13; b) expressing said first and second DNAsequences; and c) administering a molecule that binds the COI1/JA-Ilebinding pocket of COI1 to said host animal, wherein administration ofsaid molecule that binds the COI1/JA-Ile binding pocket of COI1 resultsin degradation of said target protein.
 18. The method of claim 17,wherein said host animal is a mouse.
 19. The method of claim 17, whereinsaid Arabidopsis protein COI1 or a homolog thereof is selected from thegroup consisting of Arabidopsis thaliana COI1 (SEQ ID NO:15),Arabidopsis lyrata COI1 (SEQ ID NO:16), Oryza sativa COI1 (SEQ ID NO:17), Solanum lycopersicum COI1 (SEQ ID NO: 18), Vitis vinifera COI1 (SEQID NO: 19), Populus trichocarpa COI1 (SEQ ID NO:20 and/or SEQ ID NO:21),Ricinis communis COI1 (SEQ ID NO:22), Zea mays COI1 (SEQ ID NO:23),Hevea brasiliensis COI1 (SEQ ID NO:24), Pisum sativum COI1 (SEQ IDNO:25), Nicotiana attenuate COI1 (SEQ ID NO:26), Glycine max COI1 (SEQID NO:27), Sorghum bicolor COI1 (SEQ ID NO:28), wheat COI1, andPhyscomitrella patens COI1.
 20. The method of claim 17, wherein saidmolecule that binds the COI1/JA-Ile binding pocket of COI1 is coronatineor a jasmonic acid-amino acid conjugate.
 21. The method of claim 20,wherein said jasmonic acid-amino acid conjugate is JA-Ile.
 22. Themethod of claim 17, further comprising the step of introducing aninositol pentakisphosphate cofactor into one or more cells of saidanimal.
 23. The method of claim 17, wherein said first and second DNAsequences are introduced into said animal by introduction of embryonicstem cells comprising said first and second DNA sequences during theblastocyst stage.
 24. The method of claim 17, wherein said first andsecond DNA sequences are introduced into said animal via a non-plantcell comprising said first and second DNA sequences. 25-30. (canceled)